RNA polymerase II transcription in eukaryotes requires a class of proteins called general transcription factors (Maldonado et al., Curr. Opin. Genet. Dev., 7:352-361, 1995). These proteins enable RNA polymerase II to recognize gene promoters and participate in the assembly of preinitiation complexes. One of these proteins, TFIIB, provides a physical link between the TATA-binding protein (TBP) and RNA polymerase II within the preinitiation complex (Barberis et al., Proc. Natl. Acad. Sci. U.S.A., 90:5628-5632, 1993; Buratowski et al., Proc. Natl. Acad. Sci. U.S.A., 99:5633-5637, 1993). According to a previously suggested stepwise pathway (Buratowski et al., Cell, 56:549-561, 1989), the first step of preinitiation complex formation is the binding of TBP to DNA. Next, TFIIB enters the complex followed by RNA polymerase II and other general transcription factors. As an integral component of the preinitiation complex, TFIIB also interacts with at least two other general transcription factors, TFIIF and a TBP-associated factor (TAF40) (Goodrich et al., Cell, 75:519-530, 1993; Ha et al., Genes Dev., 7:1021-1032, 1993).
One of the central problems in molecular biology is to understand how transcriptional activators stimulate gene expression in eukaryotes. At the step of transcriptional initiation, activators could in principle either increase the number of preinitiation complexes by recruiting the general transcription factors or change the quality of preinitiation complexes by increasing their stability or inducing conformational changes. Many general transcription factors including TBP (Ingles et al., Nature, 351:588-590, 1991), TAFs (Goodrich et al., ibid.), TFIIA (Ozer et al., Genes Dev., 8:2324-2335, 1994), TFIIB (Baniahmad et al., Proc. Natl. Acad. Sci. U.S.A., 90:8832-8836, 1993), TFIIF (Joliot et al., Nature, 373:632-635, 1995) and TFIIH (Xiao et al., Mol. Cell. Biol., 14:7013-7024, 1994) have been shown to interact with various transcriptional activators in vitro. In particular, a series of biochemical experiments strongly suggests that TFIIB plays an important role in transcriptional activation in vitro. First, the ability of an activator to interact with TFIIB correlates with its ability to activate transcription (Lin et al., Nature, 353:569-571, 1991). In addition, mutant TFIIB molecules that are defective in interacting with activators fail to support activated transcription in vitro, while basal transcription remains unaffected (Roberts et al., Nature, 363:741-744, 1993). Finally, transcriptional activators can stabilize the step in which TFIIB joins the preinitiation complex, a step that appears slow and/or inefficient in the absence of an activator (Lin et al., Cell, 64:971-981, 1991).
Despite these biochemical experiments indicating the importance of TFIIB in transcriptional activation, it is unknown if and how TFIIB participates in transcriptional activation in living cells. Although TFIIB is highly conserved among humans, rats, i Xenopus laevis, and Drosophila melanogaster, there is only 35% amino acid identity between yeast TFIIB (yTFIIB) and human TFIIB (hTFIIB). TFIIB molecules from eukaryotes share conserved structural motifs and are expected to form similar three-dimensional structures (Bagby et al., Cell, 82:857-867, 1996; Nikolov et al., Nature, 377:119-128, 1996). The amino-terminal domain of TFIIB contains a zinc finger motif folded into a zinc ribbon structure (Zhu et al., Struct. Biol., 3:122-124, 1996). The carboxyl-terminal core domain of TFIIB is composed of two imperfect direct repeats that are similarly folded into two subdomains consisting of mainly helices (Bagby et al., ibid., Nikolov et al., ibid.). The region between these domains appears to form a flexible and extended linker region (Barbaris et al., Proc. Natl. Acad. Sci. U.S.A., 90:5628-5632, 1993). As referred to herein, the amino-terminal zinc ribbon region of yTFIIB refers to amino acid residues 1-62; the linker region refers to residues 63-123; the carboxyl-terminal core domain refers to residues 124-345; and the first repeat in the carboxyl-terminal core domain refers to residues 124-226. The BH2 helix of yTFIIB refers to residues 144-161 and BH2-BH3 refers to residues 144-182.